Non Technical Summary The filamentous ascomycete, Cryphonectria parasitica is the plant pathogenic fungus that is the causal agent of chestnut blight, a devasting disease that has essentially eliminated the American chestnut tree from its native range in North America. To understand how the virus is able to perturb development of the chestnut blight fungus.

Goals / Objectives Transmissible hypovirulence of the chestnut blight fungus, cryphonectria parasitica, is a natural biological control for this disease. Hypovirulence is caused by a double-stranded (ds) RNA virus which infects the fungus, the result of which is a reduction of sporulation and virulence expression toward chestnut by the fungus. This effective natural biological control may be a model for how other diseases may be controlled with similar genetically engineered fungal viruses. It is the objective of this work to determine how the virus is able to regulate fungal sporulation and virulence expression and to determine if this effect can be mimicked by using a fungal vector.

Project Methods Previous work has shown that the dsRNA reduces expression of specific fungal mRNAs. This reduced mRNA accumulation is probably the result of transcriptional regulation. This needs to be determined, however. Since there are a limited number of fungal gene products affected by the virus, it is the hypothesis that the virus is perturbing the regulation of coordinately regulated fungal gene products. To test this hypothesis a number of the products being regulated, and their genes, will be isolated and their regulation studies.

Progress 02/19/99 to 02/18/04

OutputsInvestigations of how the virus CHV1 is able to perturb developmental processes of the chestnut blight fungus Cryphonectria parasticia continued. Dr. Van Alfen left Texas A&M University and this research ended.

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Publications

No publications reported this period

Progress 01/01/98 to 12/31/98

OutputsWe have been investigating how the virus CHV1 is able to perturb developmental processes of the chestnut blight fungus Cryphonectria parasticia. The phenotype of virus infected strains include poor asexual sporulation, lack of pigmentation, female sterility and low virulence (hypovirulence). The virus- induced symptoms are essentially those of a perpetual juvenile or vegetative stage of growth without development occurring. Understanding the molecular basis of these symptoms and the mechanism whereby the virus is able to induce them are the objectives of our research. Such knowledge will lead to strategies for sustainable control of pathogenic fungi in situations that do not warrant large capital investments. To date we have identified and cloned a number of host genes that when down-regulated by the virus result in some of the virus caused symptoms. For instance, we have found that the fungal sex pheromones are not expressed in virus infected strains, and that
asexual sporulation is disrupted in part by the down-regulation of a fruiting body wall protein. Most of the host proteins that have been found to be perturbed by the virus are secreted proteins and three of them are secreted through the same protein processing and secretory pathway. We have found that the virus replicates on fungal vesicles that appear to be late Golgi secretory vesicles and that these vesicles contain, as cargo, one of the virus-suppressed host proteins. We have evidence that replication of the virus on these vesicles causes an accumulation of the vesicles and blocks the transport of the cargo protein.

McCabe, P.M. and N.K. Van Alfen. 1998. Effect of a virus on transport of the hydrophobin cryparin through the secretory pathway of Cryphonectria parasitica. Phytopathology 88:S59. Publication no. P-1998-0428-AMA.

Progress 01/01/97 to 12/31/97

OutputsThe hypovirus, CHV1, causes down-regulation of expression of a number of secreted gene products of its host Cryphonectria parasitica. Among these are the cell surface hydrophobin, cryparin, the Mat 1 sex pheromone, and an extracellular laccase. We have found that in addition to their common down-regulation by the virus, each of these polypeptides contain signals for processing during secretion by the endoprotease kex2p. Additionally, we have found that one of these polypeptides, the hydrophobin cryparin, is present in the membrane vesicles that support in vitro replication of the virus; these vesicles have also been found to contain the viral polymerase. The implication of these findings is that the fungal vesicles that are used by the virus for replication function normally as transport vesicles. The common kex2p processing of secreted host proteins that are affected by the virus suggests that at least some of these vesicles are involved in kex2p. We are currently
testing this hypothesis through the study of transport of cryparin in virus-infected and isogenic uninfected strains of the fungus. Evidence in support of the hypothesis has been the isolation of a glycosylated transport intermediate of cryparin from these fungal vesicles.

OutputsThe pheromone precursor genes of the fungus Cryphonectria parasitica have been cloned and sequenced. The gene organization is very similar to that of the well characterized pheromone precursor genes of the yeasts S. cerevisiae and S. pombe. These genes are expressed in a mating-type specific manner, with only one set of the pheromone precursor genes being expressed in each mating type. These genes are among those that are down-regulated in hypovirus-infected strains of this fungus. The repression of these genes by the virus is probably responsible for the perturbation of sexual reproduction in virus infected strains. Reports that heterotrimeric G protein concentrations may be affected by the virus led us to test virus-infected strains for levels of expression of the Galphai subunit that has been claimed to be repressed in virus-infected strains. Using the pertussis toxin catalyzed ribosylation assay for Galphai subunits, we were unable to detect differences in the
amounts of this subunit between normal and virus-infected strains of the fungus. Under the conditions of growth used, the pheromone precursor genes were transcriptionally down-regulated in virus infected strains, but were normally expressed in noninfected strains. These results suggest that this Galpha subunit is not involved in the virus-caused down-regulation of the pheromone precursor genes.

OutputsThe virus responsible for transmissible hypovirulence of the chestnut blight fungus reduces the virulence and sporulation of its fungal host by repressing expression of a number of fungal genes. It isn't known how the virus is able to repress these genes, but it is becoming clear that some of them are linked in normal regulatory cascades. One of the best characterized regulatory systems of fungi is that related to sexual sporulation. We have found that the virus of the chestnut blight fungus represses expression of mating type specific genes of this fungus. The particular genes repressed by the virus that we have cloned and identified are the sex pheromone genes of this fungus. From what is known about other fungal systems, the pheromone genes are probably regulated by the mating type genes. The sex pheromone genes of Cryphonectria parasitica are similar in structure and organization to those found in S. cerevisiae. The pheromone precursor gene expressed by the "A"
mating type of the fungus suggests that the pheromone is a small prenylated peptide. There are two genes that encode this pheromone precursor. The other mating type ("a") of the fungus expresses a pheromone precursor gene transcript that encodes sever copies of a decapeptide. The virus represses expresion of both pheromone precursor genes.

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Publications

Progress 01/01/94 to 12/30/94

OutputsThe virus that causes transmissible hypovirulence of the chestnut blight fungus,Cryphonectria parasitica affects the ability of this fungus to sporulate and to attack chestnut trees. We have been investigating the molecular basis for the effects of the virus on fungal sporulation. We have recently found that the virus represses host sexual sporulation by transcriptional down-regulation of fungal mating-type-specific genes. We have cloned genes from both mating types of the fungus that are specifically repressed in viral infected strains. These genes are only expressed in one of the mating types of the fungus and are thus thought to play a role in the mating process. One of these genes, Vir2, has been deleted, with the null mutant have a phenotype that mimics part of the symptom complex caused by the virus, including sexual sterility. The sequence of this gene, and another one, Vir1, contain the sequences conserved in all known prenylated fungal pheromones. There is
evidence that repression of fungal sexual reproduction enhances the ability of the virus to move within populations of its host. One graduate student and a post-doctoral student worked on this project.

OutputsTransmissible hypovirulence has been found to effectively control chestnut blight. This biological control is the result of a double-stranded (ds)RNA virus which moves through the population of the chestnut blight fungus causing the fungus to exhibit reduced sporulation and virulence. We have been investigating the mechanism of virus replication and how the virus is able to reduce fungal virulence and sporulation. We have found that the virus replicates in a manner similar to that reported for many plus-sense single-strand RNA viruses. The virus replicates in association with host membranes. We recently isolated an active replication complex from these host membranes and identified the putative viral encoded RNA polymerase among the proteins of the complex using antibody made against a polypeptide expressed from the conserved RNA polymerase region of the viral genome. In our efforts to understand the effect of the virus on host sporulation, we have been able to mimic
some of the symptoms of viral infection by specific deletion of one of the viral affected host genes. The mutation confirmed that the gene encodes a host gene which affects both asexual and sexual sporulation. We are now seeking genes that regulate the expression of this sporulation gene. Two graduate students and a post-doctoral student worked on this project.

VAN ALFEN, N.K. 1994. Molecular approach to understanding symptoms caused by a virus of Cryphonectria parasitica. In: The proceedings of the International Workshop on Viruses of Fungi and Simple Eukaryotes, Yigal Koltin and Victor Rubio,.

OutputsMany fungal plant pathogens are very difficult to control using current technology. Fungal viruses offer potential for control of some of these diseases, particularly under circumstances where chemical controls cannot be used. The greatest potential is in using these viruses as vectors for recombinant genes which can disrupt normal fungal developmental processes. In our studies toward this goal, we have identified genes of the fungal forest pathogen, Cryphonectria parasitica repressed in the presence the virus responsible for transmissible hypovirulence. The genes we have identified and cloned appear to be primarily involved in sporulation. We have found that one of the cloned genes is necessary for sexual reproduction, and probably encodes a fungal pheromone. The transcription of each of these genes is repressed at the transcriptional level in the presence of the virus as determined by nuclear run-on assays. We are currently using genetic approaches to identify and
clone genes which regulate the normal expression of these four maker genes. Two graduate students and a post-doctoral student worked on this project.